Burning seeds: Germinaton and resprouting by Juli Pausas

Journal of Ecology 2008, 96, 543–552

doi: 10.1111/j.1365-2745.2008.01359.x

Burning seeds: germinative response to heat treatments in relation to resprouting ability

Blackwell Publishing Ltd

S. Paula and J. G. Pausas* Centro de Estudios Ambientales del Mediterráneo (CEAM), Charles R. Darwin 14, Parc Tecnològic, Paterna, València 46980, Spain

Summary 1. In Mediterranean fire-prone ecosystems, plant species persist and regenerate after fire by resprouting, by recruiting new individuals from a seed bank (post-fire seeding), or by both resprouting and post-fire seeding. Since species with resprouting ability are already able to persist in fire-prone ecosystems, we hypothesize that they have been subjected to lower evolutionary pressure to acquire traits allowing or enhancing post-fire recruitment. Consequently, we predict that the germination of non-resprouters is more likely to be increased or at least unaffected by heat than the germination of resprouters. 2. To test this hypothesis we compiled published experiments carried out in Mediterranean Basin species where seeds were exposed to different heat treatments. We compared the probability of heat-tolerant germination (i.e. heated seeds had greater or equal germination than the control), the probability of heat-stimulated germination (i.e. heated seeds had greater germination than the control) and the stimulation magnitude (differences in proportion of germination of the heated seeds in relation to the untreated seeds, for heat-stimulated treatments) between resprouters and non-resprouters. 3. Non-resprouters showed higher probability of heat-tolerance, higher probability of heatstimulation and higher stimulation magnitude even when phylogenetic relatedness was considered. Differences between life-forms and post-fire seeding ability do not explain this pattern. 4. Non-resprouters appear to have a greater capacity to both (i) persist after fire by means of recruiting (greater heat-tolerance) and (ii) increase their population after fire (greater heat-stimulated germination), than resprouters. 5. Synthesis. Our results contribute to understanding the factors that condition the evolution of fire-persistence plant traits and support the hypothesis that resprouting and post-fire recruitment are negatively associated in Mediterranean Basin flora. Key-words: fire-stimulated germination, heat treatments, Mediterranean Basin, resprouters, seeders

Introduction Fire is one of the most predictable disturbances faced by plants in Mediterranean ecosystems (Di Castri et al. 1981). Plant species have two basic mechanisms for persisting in Mediterranean fire-prone ecosystems (Keeley & Zedler 1978; Pausas et al. 2004): by regenerating their above-ground biomass (i.e. resprouting) by plants that survived the fire (persistence at the individual and population-level) and by recruiting new individuals from a fire-resistant seed bank (persistence at the population-level only). Resprouters and seeders are the typical terms used to refer to the species that

*Correspondence author. E-mail: pausas@gmail.com; juli@ceam.es

regenerate after fire primarily through the first or the second mechanism, respectively (Keeley & Zedler 1978). In fireprone ecosystems, resprouters and seeders coexist; in addition, many species may have traits that allow them to both resprout and recruit after fire (e.g. Gill 1981; van Wilgen & Forsyth 1992; Enright & Goldblum 1999; Pausas et al. 2006). In the Mediterranean Basin, there is a negative correlation between the traits associated with post-fire resprouting and those associated with post-fire seeding such that post-fire seeding traits appeared later in evolution than resprouting traits and were mainly acquired by non-resprouting lineages (Pausas & Verdú 2005). These results suggest that both strategies may be efficient for persistence in Mediterranean fire regimes and that having one of them may reduce the probability of acquiring the other.

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Post-fire recruitment may succeed because the heat of the fire may break seed dormancy or quiescence, and enhance germination (e.g. Thanos et al. 1992; Herranz et al. 1998; Keeley & Fotheringham 2000). Increased permeability of the seed coat and, to a lesser extent, induction of physiological processes and denaturing of seed coat inhibitors, are implicated in fire-triggered seed germination (Keeley 1991; Thanos et al. 1992; Bell et al. 1993; van Staden et al. 2000). Furthermore, in some species, seeds resist the heat produced by fire, but germination is not fire-stimulated (Trabaud & Casal 1989). Other germination cues related to fire, such as smoke, charcoal and nitrogenous compounds, have also been identified (Keeley & Bond 1997; Keeley & Baer-Keeley 1999; Clarke et al. 2000; van Staden et al. 2000; Wills & Read 2002; Pérez-Fernández & Rodríguez-Echevarría 2003); nevertheless, to respond to any of these cues, seeds must first survive the high temperatures released during a fire. Fire-stimulated germination is common in most Mediterranean-climate shrublands although it seems to be taxonomically aggregated, most common in the Cistaceae and Fabaceae, and in some clades of the Rhamnaceae and Malvaceae (Thanos et al. 1992; Bell et al. 1993; Keeley & Bond 1997; Keeley & Fotheringham 2000). Nevertheless, it has been suggested that this trait is less frequent in the Mediterranean Basin than in other Mediterranean ecosystems (Keeley & Baer-Keeley 1999), and a substantial amount of post-fire recruitment is probably the result of the seeds being tolerant to fire rather than being stimulated by it (Buhk & Hesen 2006; Luna et al. 2007). Both fire-resistant and fire-stimulated germination require the presence of a persistent seed bank or the production of heat-resistant seeds just before fire (Pausas et al. 2004). Dormancy, which is strongly correlated with persistence of seed banks (Thompson et al. 1998), is a widespread heritable trait among angiosperms (Baskin et al. 2000). However, great inter- and intra-variability in seed dormancy and heatstimulated germination has been found in several species (Keeley 1991; Pérez-García 1997; Herranz et al. 1999; Cruz et al. 2003a; see Tieu et al. 2001 for variability in the germinative response to smoke). Because natural selection acts on the variability of heritable traits, the evolution of heat-stimulated germination could be determined by the intensity of the selective pressure. Whereas the resistance of seeds to fire only ensures the persistence of the plant population, fire stimulated germination permits a large number of offspring to be produced after fire and increases the population size compared with the pre-fire population (Roy & Sonie 1992; Ladd et al. 2005). Rapid post-fire recruitment enhances plant fitness by accessing more resources (Bond & van Wilgen 1996) and thus growing faster, shortening the time to reach maturity after fire and increasing the probability of storing a large amount of seeds before the next fire (Le Maitre & Migdley 1992; Verdú & Traveset 2005). Consequently, post-fire seedling emergence is under strong selective pressure. We hypothesize that because species with resprouting ability are able to persist in fire-prone ecosystems, they may

have been subjected to lower evolutionary pressure to acquire traits that enable or enhance their recruitment after fire. On the contrary, species with no resprouting ability can only persist in fire-prone environments if they have an efficient post-fire recruitment process. Consequently, we expect a higher germinative response to heat treatments in non-resprouters than in resprouters. That is, we predict that the germination of non-resprouters is more likely to be increased or at least unaffected by heat than the germination of resprouters. To test this hypothesis we performed an exhaustive compilation of published experiments in which seeds from Mediterranean Basin species were exposed to different heat treatments (combinations of temperature and exposure time), and then we compared the germinative response of the resprouters and non-resprouters. The germinative response was characterized as: (i) heat-tolerance, if germination of the heated seeds was greater or equal to that of the control; and (ii) heat-stimulation, if germination of heated seeds was greater than that of the control. The first case is related to the potential persistence of the population regardless of changes in population size, while the second indicates a potential increase in the post-fire population size. So, specifically, we tested whether heattolerance and heat-stimulation are higher for non-resprouters than for resprouters.

Methods DATA SET

We compiled a data set on heat experiments, that is, experimental germination tests under different temperatures and exposure times, performed on species of the Mediterranean Basin. We considered only dry-heat exposure experiments (cases using scarification with boiling water were not included) and only those in which the temperatures applied were ≥50 °C. For each study and species, we compiled the percent germination of the control and the percent germination of each heat treatment (combination of temperature and exposure time). Cases in which both the control and the treatment showed 0% germination were excluded. For experimental designs in which seeds of the same species and population were subjected to different storage conditions (e.g. with and without stratification) and/or incubation regime (e.g. in dark and in light conditions), we included only the germination test in which the untreated seeds showed the highest germination. This is a conservative criterion for avoiding pseudoreplication in the data set. Experiments performed on seeds from serotinous species were discarded because seeds inside cones are protected from the heat of a fire (Habrouk et al. 1999). In the Mediterranean Basin, serotiny is only found in some conifers (Pausas 1999), and the amount of serotinous cones varies greatly among populations (Goubitz et al. 2004; Tapias et al. 2004); furthermore, the selective pressure of fire on serotinous species may act on different traits from seed traits (e.g. cone traits). To ensure consistency, all coniferous species were excluded from the data set; our analysis was thus restricted to angiosperms. For the compilation, species names were standardized following the European Science Foundation – European Documentation System (ESFEDS) which is largely based on Flora Europaea (Tutin et al. 1964–80). The ESFEDS database was checked in the Global Plant Checklist of the International Organization for Plant Information (IOPI 1996–2005). Other criteria were followed in cases where

Post-fire germination and resprouting 545 some important taxonomical updates had been carried out (e.g. Talavera et al. 1999 for Genisteae) or when some species were missing in the ESFEDS database (e.g. Greuter et al. 1984–89 for Cistaceae). For family names, the Angiosperm Phylogenetic Group standards were used (Bremer et al. 2003). We found 53 references reporting appropriate data on heat experiments for 164 species or subspecies included in 28 families and 85 genera (Appendix S1 in Supplementary material). This yielded a total of 1684 entries. Most of these studies (78%) report the statistical test between the control and the treatment. For each species in the data set, we compiled its post-fire response based on bibliographic references and personal field observations. Because the germinative response to fire cues is related to the lifeform (Keeley & Bond 1997; Clarke et al. 2000; van Staden et al. 2000), we also compiled this trait, mostly from local floras. The lifeforms considered were: woody plants, perennial herbs (including hemicryptophytes, geophytes and some plants slightly lignified at the base) and annuals. We considered that a species had the ability to resprout (R+) if there was field evidence of resprouting after 100% scorching (Gill 1981; Pausas et al. 2004); otherwise, it was considered a non-resprouter (R–). Species with field evidence of post-fire seedling emergence from a soil seed bank or from seeds produced just before fire were considered seeders (S+); otherwise they were considered non-seeders (S–). Note that species recruiting after fire from outside populations (arriving by dispersal) were not regarded as S+ as their seeds are not subjected to fire. Therefore, four post-fire strategies are possible: R+S–, R+S+, R–S+ and R–S– (Pausas et al. 2004). Life-form was assigned to all species, but resprouting and post-fire seeding was obtained for 120 and 105 species, respectively; thus, when testing the resprouting or seeding factor, the total cases used were lower (1399 and 1451, respectively). In general, most species were woody plants (61%), with similar frequency among R+ and R–, whereas most of the perennial herbs were R+. S+ species were more abundant than S– and their frequency was higher among R– than among R+ (80% and 41%, respectively).

ANALYSIS

Heat experiments involve very different combinations of temperature and exposure time. To estimate the accumulated heat dose received by the seeds in each treatment, we multiplied the temperature (in degrees Celsius) by the natural logarithm of the exposure time plus one (in minutes). This heat index (H) is analogous to the concentrationtime product used in toxicology (e.g. Wang et al. 2004 and references therein). In the compiled data, essayed temperatures ranged from 50 to 300 °C, exposure times ranged from 1 min to 24 h and the heat index (H) ranged from 34.7 to 727.3 units (Fig. 1). For each treatment, the germinative response was first classified as greater, equal or lower than the control (i.e. stimulated, unaffected or inhibited by heat, respectively) on the basis of the significance of the statistical test provided in each study. A set of cases (22%) did not include any statistical test. These cases include both resprouting and non-resprouting species (51% and 49%, respectively). In order to avoid any loss of information, we classified the germinative response of statistically non-tested cases by comparing them with the tested experiments. For the statistically tested experiments, we plotted the frequency distribution (and the smoothed density distribution) of the difference in percent germination between control and treatment for the cases with greater, equal and lower germination than the control. The intersections between the three smoothed density distributions were –13 and 14. We then used these thresholds to classify the statistically non-tested treatments as stimulated

Fig. 1. Relationship between temperature and exposure time for different heat index (H) values (contour lines).

(differences in percent germination between control and heated seeds higher than 14%), inhibited (differences lower than –13%), or unaffected (in between) germination. If we apply these thresholds to the statistically tested treatments we observe that the percentage of misclassified cases is 10%. This classification error was mainly (6.9%) due to the failure to detect a significant difference when it was different (type II error or false negative); therefore, the classification is conservative. Furthermore the frequency of treatments that stimulate, do not affect or inhibit germination was not significantly different between the tested and non-tested treatments, both when considering all species (χ2 = 0.54, d.f. = 2, P = 0.762) or each resprouting ability separately (R+: χ2 = 4.99, d.f. = 2, P = 0.083; R–: χ2 = 0.67, d.f. = 2, P = 0.715). The close-to-significant χ2-test for R+ is due to a slight over-representation of inhibited germination cases in the statistically non-tested data set, and thus caution should be taken only if the results comparing heat-tolerance between resprouting abilities (see below) were not strongly significant. It is notable that the whole data set includes more treatments (29%), more species (8%) and more treatments per species (19%) than the tested subset, and a wider overall range of heat index (34.7–727.3 for the whole data set vs. 34.7–456.7 for the tested treatments). The probability of heat-tolerance (i.e. of having greater or equal germination than the control) and heat-stimulation (i.e. of having greater germination than the control) was analysed using a GLM model with a binomial error distribution and logit link function, and tested by an analysis of deviance (McCullagh & Nelder 1989). To analyse the extent to which germination was different between resprouting abilities, we used resprouting as a factor and the heat index (H) as a covariable. Since all life-forms and post-fire seeding abilities were not equally represented in the data set between R+ and R– (see above), we also compared the probability of tolerance and stimulation between life-forms and between post-fire seeding abilities (S). We expect a decreasing probability of heat-tolerance with H and a unimodal model of heat-stimulated germination probability in relation to H (i.e. low germination at low and high intensities); thus, we tested a monotonic response of heat-tolerance to H and a quadratic response of heat-stimulation to H (i.e. H + H2). In all

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analyses, species was also included in the model, nested in either post-fire regeneration abilities or life-form. For treatments that produced stimulation, we evaluated to what extent the heat treatment increased germination in a different manner between R+ and R–, by testing the differences in the proportion of seeds germinating between the heated and the control treatments (hereafter, stimulation magnitude) against H and resprouting using the analysis of variance. As for the stimulation probability, we assumed a unimodal model in relation to H and included species as a factor nested in resprouting. Because both resprouting and post-fire seeding are phylogenetically structured in the Mediterranean Basin (Herrera 1992; Pausas & Verdú 2005; Verdú & Pausas 2007), our differences in germination response between resprouting abilities could be driven by species relatedness. To evaluate this, we performed a test of the effect of resprouting ability on the probability of heat-tolerance and heat-stimulation, and on the stimulation magnitude, considering the phylogenetic relatedness among the species in our data set. We first assembled a phylogenetic tree with branch lengths for our species with the help of the Phylomatic software implemented in P 3.41 (Webb et al. 2007) and using the angiosperm megatree version R20050610.new. Then, we classified all the studied heat treatments in the following H classes: very low (H < 100), low (100 < H < 200), medium (200 < H < 300), high (300 < H < 400) and very high (H > 400). To conduct the phylogenetic analyses we recalculated the three dependent variables as follows: for each species and each H class we counted the cases with successful heat-tolerance and heatstimulation, and computed the average magnitude. Finally, for each H class we tested the effect of resprouting ability on each of the dependent variables using a generalized estimating equation and including the phylogenetic relatedness among species (obtained from the phylogenetic tree) as a correlation matrix in the model (see Paradis & Claude 2002). As in the non-phylogenetic analyses, we assumed a binomial error distribution and logit link function for heat-tolerance and heat-stimulation, and a normal error distribution for magnitude. The advantages of using generalized estimating equation rather than other comparative methods is that it permits the use of qualitative variables (such as resprouting ability), the presence of polytomies in the phylogenetic tree, and the use of binomial error distributions (for germination probabilities). This analysis was performed using the  software (Paradis et al. 2004).

Results HEAT-TOLERANCE: GERMINATION GREATER THAN OR EQUAL TO THE CONTROL

The probability of seed heat-tolerance was very high (close to 1) at low H values, decreasing with H either progressively or suddenly depending on the life-form and the post-fire regeneration strategy (Fig. 2). The probability of heat-tolerance was higher for R– species that for R+ ones (Fig. 2a). Both the H and its interaction with resprouting ability were significant (Table 1), suggesting that the probability of heat-tolerance decreases with H, and the decreasing pattern was quicker for R+ than for R– species (Fig. 2a). Considering the phylogeny, there were no differences between R+ and R– for the very low and low H classes (P = 0.484 and P = 0.207, respectively), but differences were significant and marginally significant for the moderate and high H class (P = 0.039 and P = 0.053,

Fig. 2. Tolerance probability in relation to heat treatments for the different resprouting abilities (a, d), life-forms (b) and post-fire seeding abilities (c). In figure d, only woody S+ species were considered. Fits include significant (P < 0.05) effects only (see Tables 1 and 2 for further statistical details). Dotted lines refer to SEs of the fitted line.

respectively). Nevertheless, differences between resprouting abilities for the very high H class were non-significant (P = 0.109) and thus attributable to the phylogenetic arrangement of the species at the higher end of the H gradient. Differences between life-forms were also significant, with perennial herbs showing the lowest probability of heat-tolerance (Table 1, Fig. 2b). The interaction between life-form and H was also highly significant (Table 1): woody plants and annuals showed some degree of tolerance even at high H, whereas for perennial herbs tolerance decreased rapidly (Fig. 2b). S+ showed a higher heat-tolerance probability than S–, although the pattern of tolerance probability with H was quite similar for both S+ and S– (no significant interaction; Table 1, Fig. 2c). Considering all these results, the previous differences detected between resprouting abilities could be due to the fact that, in our data set, S– and perennial herbs were more frequent in R+ species than in R– (see Methods) and both had a low heat-tolerance probability (Table 1, Fig. 2b and c). However, when this comparison was restricted to woody S+ species (i.e. comparison between R+S+ and R–S+), we still detected differences between R+ and R–, although for high H values only (significant resprouting ability–H interaction; Table 2, Fig. 2d). HEAT-STIMULATION: GERMINATION GREATER THAN THE CONTROL

The probability of heat-stimulated germination was different between resprouting abilities (Table 3). R– showed a higher

Post-fire germination and resprouting 547 Table 1. Analysis of deviance for the probability of heat-tolerant germination in relation to resprouting ability, life-form, seeding ability and heat index

Source of variation

d.f.

Deviance

Null Resprouting (R) Heat index (H) R×H Species in R Explained deviance (%)

1 1 1 118

8.80 147.79 12.38 321.77 41.47

Null Life-form (LF) Heat index (H) LF × H Species in LF Explained deviance (%)

2 1 2 167

8.18 165.09 13.51 438.84 43.59

Null Seeding (S) Heat index (H) S×H Species in S Explained deviance (%)

1 1 1 104

21.66 132.22 0.36 283.22 37.13

Residual d.f.

Residual deviance

1398 1397 1396 1395 1277

1183.26 1174.46 1026.67 1014.29 692.52

** **** *** ****

1683 1681 1680 1678 1511

1435.43 1427.24 1262.15 1248.64 809.79

* **** ** ****

1450 1449 1448 1447 1343

1178.32 1156.66 1024.44 1024.09 740.87

**** **** 0.55 ****

All species are included in the analysis. (Levels of significance: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; P values are shown when P ≥ 0.05).

Table 2. Analysis of deviance for the probability of heat-tolerant germination in relation to resprouting ability and heat index considering only woody S+ species

Source of variation

d.f.

Deviance

Null Resprouting (R) Heat index (H) R×H Species in R Explained deviance (%)

1 1 1 52

0.66 90.29 11.82 143.41 31.24

Residual d.f.

Residual deviance

1055 1054 1053 1052 1000

787.92 787.26 696.97 685.15 541.75

0.42 **** *** ****

(Levels of significance: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; P values are shown when P ≥ 0.05).

stimulation probability than R+ (Fig. 3a). Moreover, the relationship between stimulation probability and H differed between them (Table 3): for low H values, the stimulation probability increased with H at a similar rate in R+ and R–, reaching their respective maximum values of probability for slightly different H values; stimulation was negatively affected by H sooner in R+ than in R–, in such a way that for very high H values, no R+ species was stimulated whereas some Cistus species (R–) were still stimulated (Fig. 3a). This pattern was mostly maintained when phylogeny was considered: differences in heat-stimulated probability between R+ and R– were detected for very low, low and moderate H classes (P = 0.0024, P < 0.0001, P = 0.0027, respectively), but they disappeared for high H (P = 0.334), indicating that such differences in high H values are attributable to phylogeny and not to resprouting ability. In fact, this was expected as the phylogenetic affiliations of the data in the high H class differed between R+ (Ancardiaceae and Rosaceae) and R– (Cistaceae and Fabaceae). This comparison was not conducted for very high H, because only R– species were stimulated, all of them pertaining to the genus Cistus.

The heat-stimulation probability was higher for woody plants than for herbs, and slightly higher for annuals than for perennial herbs (Table 3, Fig. 3b). The pattern followed by the stimulation probability with H was the same for all lifeforms (non-significant life-form–H interaction; Table 3, Fig. 3b). As expected, S+ species showed a higher stimulation probability than S– ones, although the maximum stimulation probability was always < 0.4; the stimulation probability for S– was < 0.1 (Fig. 3c). The interaction between seeding ability and H was not significant (Table 3, Fig. 3c). To discount whether the differences between resprouting abilities in the probability of heat-stimulated germination were due to the heterogeneous distribution of life-forms and seeding abilities between R+ and R–, we repeated the comparison but considered only woody S+ species. For this data subset, differences between R+ and R– were detected and the interaction between resprouting ability and H remained highly significant (Table 4, Fig. 3d). The magnitude of the stimulation differed between resprouting abilities (Table 5), being higher for R– (R–: 39.7 ± 19.9 and R+: 31.8 ± 18.3, expressed as mean ± SD). The magnitude

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Source of variation

d.f.

Deviance

Null Resprouting (R) Heat index (H + H2) R × (H + H2) Species in R Explained deviance (%)

1 2 2 118

45.66 41.09 11.90 357.28 28.35

Null Life-form (LF) Heat index (H + H2) LF × (H + H2) Species in LF Explained deviance (%)

2 2 4 166

48.28 45.49 3.86 441.88 28.52

Null Seeding (S) Heat index (H + H2) S × (H + H2) Species in S Explained deviance (%)

1 2 2 104

42.09 53.70 2.47 293.86 22.58

Residual d.f.

Residual deviance

1398 1397 1395 1393 1275

1608.12 1562.47 1521.38 1509.48 1152.2

**** **** ** ****

1683 1681 1679 1675 1509

1891.74 1843.46 1797.97 1794.12 1352.24

**** **** 0.43 ****

1450 1449 1447 1445 1341

1736.96 1694.87 1641.17 1638.70 1344.83

**** **** 0.29 ****

Table 3. Analysis of deviance for the probability of heat-stimulated germination in relation to resprouting ability, life-form, post-fire seeding ability and heat index

All species are considered. Note that the heat index was fitted as a quadratic response. Levels of significance: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; P values are shown when P ≥ 0.05.

significant for the very low, low and moderate H classes (P = 0.0006, P < 0.0001, P < 0.0001, respectively), but nonsignificant for high H (P = 0.294), because stimulation was phylogenetically aggregated for these H values (see before). Differences between R+ and R– in the magnitude of stimulation for the very high H class were not tested because none of the R+ species was stimulated.

Discussion

Fig. 3. Stimulation probability in relation to heat treatments for the different resprouting abilities (a, d), life-forms (b) and seeding abilities (c). In figure d, only woody S+ species were considered. Fits include significant (P < 0.05) effects only (see Tables 3 and 4 for further statistical details). Dotted lines refer to SEs of the fitted line.

of stimulation was independent of H and the differences between resprouting abilities were unaffected by H (nonsignificant resprouting ability-H interaction; Table 5). In the phylogentic analyses, differences in the magnitude of heatstimulation between resprouting abilities (higher in R–) were

As predicted, the germinative response of seeds after heat exposure was greater in non-resprouters (R–) than in resprouters (R+), because R– seeds show higher heat-tolerance and heat-stimulation (the latter expressed as both probability and magnitude). These results were maintained even when phylogenetic relatedness was included in the analyses. Similar results were found in the Fynbos for smoke-stimulated germination, being more frequent in R– species (van Staden et al. 2000). In the Mediterranean Basin, R+S– and R–S+ are more frequent than could be expected by chance, even when phylogenetic relatedness is considered (Pausas & Verdú 2005). Therefore, differences between R+ and R– in the germinative response to heat could be due to differences between S+ and S–. Nevertheless, when the probability of heat-tolerance or heat-stimulation was compared in terms of S+ species only, the differences between resprouting abilities remained significant. Recently, a study carried out with a subset of the data included in our analysis found that the percent germination after heat-shock did not differ between R+ and R– (Luna et al. 2007), although germination after high temperatures (100 and 120 °C for 10 min) was higher for R–. The wider spatial and taxonomic range of our data set and the great amount of combinations of temperature and exposure time tested in our

Post-fire germination and resprouting 549 Table 4. Analysis of deviance for the probability of heat-stimulated germination in relation to resprouting ability and heat index, considering woody S+ species only

Source of variation

d.f.

Deviance

Null Resprouting (R) Heat index (H + H2) R × (H + H2) Species in R Explained deviance (%)

1 2 2 52

16.14 46.11 12.70 173.92 18.60

Residual d.f.

Residual deviance

1055 1054 1052 1050 998

1337.28 1321.14 1275.13 1262.43 1088.51

**** **** ** ****

Note that the heat index was fitted as a quadratic response. Levels of significance: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; P values are shown when P ≥ 0.05.

Table 5. Analysis of variance for the difference in percent germination (for treatments that produced stimulation) in relation to resprouting ability and heat index

Source of variation

d.f.

Residual d.f.

Residual SS

Null Resprouting (R) Heat index (H + H2) R × (H + H2) Species in R

1 2 1 55

4850 543 551 42 470

365 364 362 361 306

142 012 137 162 136 619 136 068 93 598

15.86 0.89 1.80 2.53

**** 0.41 0.18 ****

Note that the heat index was tested as a quadratic response. Levels of significance: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; P values are shown when P ≥ 0.05.

study increase the power of the analyses and thus highlight the differences between resprouting abilities. As a result of post-fire germination, R– species have a higher potential than R+ species to persist and even to increase their population size after fire. Moreno & Oechel (1991a) found a Californian example in which the post-fire emergence of an obligate seeder (R–S+; Ceanothus greggii) was higher than for a facultative seeder (R+S+; Adenostoma fasciculatum), especially after high-intensity fires. The authors proposed differences in the germinative response and in the seed burial depth in the soil as possible explanations for this finding. Our results support the first hypothesis, although the latter cannot be discarded. Similar results have also been found in serotinous species from Western Australia (Enright & Lamont 1989). Nevertheless, effective post-fire recruitment also depends on the amount of seed availability and seedling survival. Regarding seed availability, obligate resprouters (R+S–) typically produce fewer seeds than obligate seeders (R–S+), although the differences between R+ and R– in seed production do not always occur in S+ species (Pausas et al. 2004). In the Mediterranean Basin, species with both resprouting and seeding ability are less frequent than species that persist by means of only one of the recovery mechanisms (Pausas & Verdú 2005); therefore, differences in seed production between R+ and R– are expected, with the latter being higher (Pausas et al. 2004). Moreover, congeneric comparisons of three co-occurring R+ and R– pairs have suggested that viable seed density in the soil is higher for R– species (Kelly & Parker 1990; see Enright & Lamont 1989 for similar results in serotinous species). On the other hand, seedling establishment is the most critical phase in the life-history of Mediterranean

species, mostly limited by seasonal dryness (Mejías et al. 2002). Seedling survival under seasonal water stress is frequently higher for R– than for R+ species (Keeley & Zedler 1978; Zammit & Westoby1987; Davis et al. 1998; Enright & Goldblum, 1999). In fact, at least at leaf level, R– species show higher potential for structural resistance to drought and higher water-use efficiency than R+ (Paula & Pausas 2006). In conclusion, differences in seed availability, post-fire germination and seedling survival indicate that effective recruitment is higher in R– than in R+. The magnitude of the stimulation was independent of the heat index. Hence seed populations are quite homogeneous in relation to their heat-sensitivity, showing both refractory and non-refractory seeds (sensu Keeley 1991), but virtually no seeds with intermediate heat resistance. Thus, a pulse of germination is produced only when refractory seeds are exposed to a certain heat threshold, and the amount of germinated seeds depends on the proportion of refractory seeds, which is higher for R– species. Moreover, this heat threshold is different between resprouting abilities, since the probability of heat-stimulation was significantly affected by H. Indeed, at high H values, the probability of heat-stimulation decreases quicker in R+ than in R–, and the maximum probability of heat-stimulated germination is reached at slightly higher H values for R–. The relationship found between heat-stimulated germination and life-form is consistent with the results obtained from other fire-prone ecosystems (Keeley & Bond 1997; Clarke et al. 2000; Keeley & Fotheringham 2000). Differences between life-form have also been found for other fire cues, such as smoke or charred wood (Keeley & Bond 1997; van

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Staden et al. 2000). We found the highest probability of heattolerant and heat-stimulated germination in woody plants and the lowest in perennial herbs, whereas the annuals showed an intermediate position. A predominance of species with refractory seeds has been found in the Californian chaparral and in the South African Fynbos (Keeley & Bond 1997). In these ecosystems, annuals are frequently stimulated by heat and, in many cases, also by charred wood or smoke, which are germination cues more specifically linked to fire (Keeley 1991; Keeley & Bond 1997; Keeley & Fotheringham 2000). In the Mediterranean Basin, annuals are not necessarily linked to burned sites and tend to colonize sites recently affected by any types of disturbance (Bonet & Pausas 2004; Buhk & Hensen 2006). Furthermore, the generation time of annuals is much shorter than the fire intervals in the Mediterranean Basin, and thus fire may represent a low selective pressure for these species. Consequently, the relatively low fire-enhanced germination that we found may be expected. Nevertheless, annuals are poorly represented in the data set (they represent only 10% of the species and 6% of the treatments) and, thus, future experiments with annuals may throw further light in this regard. Perennial herbs showed the lowest probability of heatstimulation and heat-tolerance. A possible explanation is that perennial herbs are normally resprouters (only 9% were R–), and their fire-germinative response is thus low (see comparison between R+ and R–). The mean frequency of heat-stimulation for perennial herbs is also lower than for woody plants when only R+ are considered (4 ± 14% for perennial herbs and 19 ± 24% for woody plants, expressed as mean ± SD). This low germinative response of perennial herbs to heat has been found in other fire-prone ecosystems (Keeley & Bond 1997; Clarke et al. 2000), and both the longevity of the seed bank and grazing pressure have been adduced to explain it (Clarke et al. 2000). As in the Californian chaparral and the Fynbos (Keeley & Bond 1997), heat-stimulated germination in the Mediterranean Basin is quite common: of the 164 taxa included in our data set, 41% were heat-stimulated in at least one treatment and 28% in at least 25% of the treatments. Nevertheless, fire-stimulated germination appears to be phylogenetically aggregated in the Mediterranean Basin: stimulation was detected in 39% of the families, and 60% of the treatments that produced stimulated germination corresponded to a single family, the Cistaceae. Moreover, the few species stimulated at high H values were also Cistaceae. These results explain the high phylogenetic and phenotypic clustering found in communities under high fire frequency (Verdú & Pausas 2007; Pausas & Verdú, in press). We are aware that our data set is biased, and that the species for which heat-stimulated germination is expected were over-represented (e.g. 39% of the treatments were carried out with Cistaceae). Nevertheless, the broad taxonomic and phylogenetic spectrum included in this data set suggests that any possible bias in selection of the species studied may also represent the taxonomic aggregation of the heat-stimulated germination trait. This aggregation has been explained by the relationship between germination

patterns and seed anatomy, with the latter being highly conserved at high taxonomic levels (see Keeley & Bond 1997 and references therein). Our results highlight the importance of heat intensity for understanding post-fire germination; because it also affects resprouting ability (Moreno & Oechel 1991b; Lloret & LópezSoria 1993; Cruz et al. 2003b), it is a major factor in the postfire recovery of Mediterranean Basin ecosystems. In fact, germination ability of seeds in response to heat is missing in most global trait databases (e.g. Cornelissen et al. 2003) and could be a valuable trait to improve the quality and the predictive value of such compilations. Nevertheless, other fire-related factors may also stimulate seed germination (e.g. smoke, charred wood). These other germination cues have not yet been studied extensively and, at present, they seem to be relatively unimportant in the Mediterranean Basin (Keeley & Baer-Keeley 1999; Pérez-Fernández & Rodríguez-Echeverria 2003; Rivas et al. 2006; Crosti et al. 2006). However, given that the differences between R+ and R– observed in this paper are similar to those observed in smoke-stimulated germination for South Africa flora (van Staden et al. 2000), we expect R– species to be stimulated more by any fire-related germination cue. Further research is undoubtedly needed to understand fully the role of these mechanisms on post-fire regeneration.

Acknowledgements We thank C. Pérez-Cervelló for his kind contribution to the database compilation, M. Arianotsou for providing access to Greek grey literature, and B. Moreira and two anonymous referees for their helpful comments and suggestions on the manuscript. This work has been financed by SINREG (REN2003-07198-C0202/GLO) and PERSIST (CGL2006-07126/BOS) projects from the Spanish Government. CEAM is supported by Generalitat Valenciana and Bancaixa.

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Zammit, C. & Westoby, M. (1987) Seedling recruitment strategies in obligateseeding and resprouting Banksia shrubs. Ecology, 68, 1984–1992. Received 10 October2007; accepted 25 January 2008 Handling Editor: Peter Bellingham

Supplementary material The following supplementary material is available for this article:

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Appendix S1 List of species considered, life-form, fire response and data sources.

Paula & Pausas - J. Ecol. - Supplementary material: Appendix S1 - 1

Appendix S1 List of species considered in this study with their life form (LF), resprouting ability (R), post-fire seeding (S) and data source of heat treatments (Sources; complete reference at the bottom of the table). AH= annual herb; PH; perennial herb; W = woody plant; NA= no reliable data. Species Family Acinos alpinus (L.) Moench ssp. meridionalis Lamiaceae (Nyman) P.W. Ball Adenocarpus lainzii (Castrov.) Castrov. Fabaceae Agrostis curtisii Kerguélen Poaceae Agrostis delicatula Pourret ex Lapeyr. Poaceae Alyssum serpyllifolium Desf. Brassicaceae Allium sp. Liliaceae Anarrhinum bellidifolium (L.) Willd. Scrophulariaceae Anthyllis lagascana Benedí Fabaceae Antirrhinum litigiosum Pau Scrophulariaceae Arbutus unedo L. Ericaceae Arenaria aggregata (L.) Loisel. ssp. erinacea Caryophyllaceae (Boiss.) Font Quer Argyrolobium zanonii (Turra) P.W.Ball Fabaceae Asterolinon linum-stellatum (L.) Duby Primulaceae Avenula marginata (Lowe) J. Holub Poaceae Betula pendula Roth Betulaceae Brachypodium retusum (Pers.) Beauv. Poaceae Briza maxima L. Poaceae Briza media L. Poaceae Calicotome intermedia C. Presl Fabaceae Calluna vulgaris (L.) Hull Ericaceae Catananche caerulea L. Asteraceae Centaurea alba L. ssp. alba Asteraceae Centaurea aspera L. ssp. aspera Asteraceae Centaurea boissieri DC. ssp. duffourii Dostál Asteraceae Centaurea boissieri DC. ssp. pinae (Pau) Dostál Asteraceae Centaurea calcitrapa L. Asteraceae Centaurea cyanus L. Asteraceae Centaurea leucophaea Jordan ssp. leucophaea Asteraceae Centaurea linifolia L. ssp. linifolia Asteraceae Centaurea maculosa Lam. ssp. albida (Lecoq & Asteraceae Lamotte) Dostál Centaurea maculosa Lam. ssp. maculosa Asteraceae Centaurea melitensis L. Asteraceae Centaurea ornata Willd. ssp. ornata Asteraceae Centaurea ornata Willd. ssp. saxicola (Lag.) Dostál Asteraceae Centaurea paui Loscos Asteraceae Centaurea pectinata L. ssp. pectinata Asteraceae Centaurea rouyi Coincy Asteraceae Centaurea scabiosa L. ssp. scabiosa Asteraceae Centaurea seridis L. Asteraceae Centaurea solstitialis L. ssp. solstitialis Asteraceae Centaurea spinabadia Bubani ex Timb.-Lagr. ssp. Asteraceae hanryi (Jordan) Dostál Centaurea uniflora L. ssp. emigrantis (Bub.) Pau et Asteraceae F.Q. Centaurea vinyalsii Sennen Asteraceae Ceratonia siliqua L. Fabaceae Cistus albidus L. Cistaceae Cistus clusii Dunal Cistaceae Cistus creticus L. Cistaceae

LF W

R NA

S NA

Sources 21

W PH PH AH PH PH W PH W W

+ + + + NA NA NA + NA

+ + NA NA NA NA NA NA

37 14, 15 15 21 20 21 31 21 22 21

W AH PH W PH AH PH W W PH PH PH PH PH PH AH PH PH PH

NA + + + + NA NA NA NA NA NA NA + NA

+ + NA + + NA NA NA NA NA NA NA NA NA

17 7 14, 15 32, 33 7 38 38 7 12, 15, 52 21 36 36 36 21 36 36 36 36 36

PH AH PH PH PH PH PH PH PH AH PH

+ NA NA NA + NA + NA NA

+ NA NA NA + NA NA NA + NA

36 36 21 36 36 36 36 36 36 36 36

PH W W W W

NA + -

NA + + +

36 2, 27 7, 9, 40, 44, 45, 53 7, 8, 18, 23, 44 20, 28, 43, 48

Paula & Pausas - J. Ecol. - Supplementary material: Appendix S1 - 2 Cistus creticus L. ssp. corsicus (Loisel.) Greuter & Burdet Cistus crispus L. Cistus ladanifer L. Cistus laurifolius L. Cistus libanotis L. Cistus monspeliensis L. Cistus palhinhae Ingram Cistus parviflorus Lam. Cistus populifolius L. ssp. populifolius Cistus psilosepalus Sweet Cistus salviifolius L.

Cistaceae

Cistaceae Cistaceae Cistaceae Cistaceae Cistaceae Cistaceae Cistaceae Cistaceae Cistaceae Cistaceae

W W W W W W W W W W

+ + + + + + + + + +

Clematis vitalba L. Convolvulus lanuginosus Desr. Crucianella angustifolia L. Cytisus multiflorus (L'HĂŠr.) Sweet Cytisus oromediterraneus Rivas Mart. & al. Cytisus scoparius (L.) Link ssp. reverchonii (Degen & Hervier) Rivas Goday Cytisus scoparius (L.) Link ssp. scoparius Cytisus striatus (Hill) Rothm. Chamaerops humilis L. Chondrilla juncea L. Daboecia cantabrica (Hudson) C. Koch Danthonia decumbens (L.) DC. Daucus carota L. Dipcadi serotinum (L.) Medicus Dorycnium pentaphyllum Scop. Elaeoselinum tenuifolium (Lang.) Lange Erica arborea L. Erica australis L. Erica ciliaris L. Erica erigena R. Ross Erica scoparia L. Erica tetralix L. Erica umbellata L. Erica vagans L. Fumana ericoides (Cav.) Gand. Fumana laevipes (L.) Spach Fumana procumbens (Dunal) Gren. & Godr. Fumana thymifolia (L.) Spach ex Webb Galium fruticescens Cav. Genista berberidea Lange Genista florida L. Genista triacanthos Brot. Gladiolus illyricus Koch Halimium atriplicifolium (Lam.) Spach. Halimium halimifolium (L.) Willk ssp. halimifolium Halimium halimifolium (L.) Willk ssp. multiflorum (Dunal) Maire Halimium lasianthum (Lam.) Spach ssp. alyssoides Greuter Halimium ocymoides (Lam.) Willk. Halimium umbellatum (L.) Spach Halimium viscosum (Wilk.) P. Silva Helianthemum apenninum (L.) Mill. Helianthemum cinereum (Cav.) Pers ssp. rotundifolium (Dunal) Greuter & Burdet

Ranunculaceae Convolvulaceae Rubiaceae Fabaceae Fabaceae Fabaceae

W W AH W W W

+ NA + + +

+ + + + +

18, 44 9, 11, 30, 49 9, 19, 25, 49 18 23, 28, 40, 44, 45, 53 44 44 18 15, 44 7, 9, 23, 41, 43, 44, 45, 48 41 7 20 1, 34 1 17

Fabaceae Fabaceae Arecaceae Asteraceae Ericaceae Poaceae Apiaceae Liliaceae Fabaceae Apiaceae Ericaceae Ericaceae Ericaceae Ericaceae Ericaceae Ericaceae Ericaceae Ericaceae Cistaceae Cistaceae Cistaceae Cistaceae Rubiaceae Fabaceae Fabaceae Fabaceae Iridaceae Cistaceae Cistaceae Cistaceae

W W W PH W PH PH PH W PH W W W W W W W W W W W W PH W W W PH W W W

+ + + NA + + NA + + + + + + NA + + + NA + + + + -

+ + NA + + + + NA NA + + NA NA + + + + + + + NA + + + + + +

37, 42 17, 34, 37 41 21 15, 51 38 21 7 17 7 13, 22, 51 10, 13, 51, 52 12, 15 12 13 13 13, 15 13, 26 7, 21 7 21 7 21 37 25, 42 37 7 7, 18 18, 44 18

Cistaceae

15, 25

Cistaceae Cistaceae Cistaceae Cistaceae Cistaceae

W W W W W

NA -

+ + + + +

18, 44 25, 52 18, 21 21 21

Paula & Pausas - J. Ecol. - Supplementary material: Appendix S1 - 3 Helianthemum croceum (Desf.) Pers. Helianthemum hirtum (L.) Mill. Helianthemum pilosum (L.) Pers. Helichrysum italicum (Roth) G. Don fil. ssp. serotinum (Boiss.) P. Fourn. Helichrysum stoechas (L.) Moench Hippocrepis ciliata Willd. Hippocrepis multisiliquosa L. Holcus lanatus L. Inula montana L. Lavandula latifolia Medicus Lavandula stoechas L. Leucanthemopsis pallida (Miller) Heywood Leuzea conifera (L.) DC. Linum narbonense L. Marrubium supinum L. Medicago arborea L. Misopates orontium (L.) Rafin Nepeta nepetella L. Ononis ornithopodioides L. Paronychia capitata (L.) Lam. Periploca laevigata Aiton ssp. angustifolia (Labill.) Markgraf Phillyrea latifolia L. Phlomis fruticosa L. Phlomis lychnitis L. Pistacia lentiscus L. Plantago sempervirens Crantz Prunella laciniata (L.) L. Pseudarrhenatherum longifolium (Thore) Rouy Psoralea bituminosa L. Pterospartum tridentatum (L.) Willk. Quercus ilex L. ssp. rotundifolia (Lam.) T. Morais Quercus pyrenaica Willd. Quercus robur L. Reseda lutea L. Reseda luteola L. Rhus coriaria L. Rosmarinus officinalis L. Salvia pratensis L. Salvia verbenaca L. Santolina chamaecyparissus L. Sarcopoterium spinosum L. Scabiosa turolensis Pau ex Willk. Scolymus hispanicus L. Scorpiurus muricatus L. Sideritis hirsuta L. Sideritis tragoriganum Lag. Silene mellifera Boiss. & Reuter Staehelina dubia L. Stipa tenacissima L. Telephium imperati L. Teucrium carthaginense Lange Teucrium expansum Pau Teucrium polium L. ssp. capitatum (L.) Arcangeli Teucrium pseudochamaepitys L. Thapsia villosa L. Thymus leptophyllus Lange Thymus mastichina L.

Cistaceae Cistaceae Cistaceae Asteraceae

W W W W

NA NA

NA + NA NA

44 21 44 21

Asteraceae Fabaceae Fabaceae Poaceae Asteraceae Lamiaceae Lamiaceae Asteraceae Asteraceae Linaceae Lamiaceae Fabaceae Scrophulariaceae Lamiaceae Fabaceae Caryophyllaceae Apocynaceae

W AH AH PH PH W W PH PH PH PH W AH W AH W W

NA + NA NA + NA + NA NA +

NA NA NA + NA + + NA NA NA NA NA + NA + NA -

21 7 16 38 21 21 20, 21 21 21 21 21 28 20 21 7 21 7

Oleaceae Lamiaceae Lamiaceae Anacardiaceae Plantaginaceae Lamiaceae Poaceae Fabaceae Fabaceae Fagaceae Fagaceae Fagaceae Resedaceae Resedaceae Anacardiaceae Lamiaceae Lamiaceae Lamiaceae Asteraceae Rosaceae Dipsacaceae Asteraceae Fabaceae Lamiaceae Lamiaceae Caryophyllaceae Asteraceae Poaceae Caryophyllaceae Lamiaceae Lamiaceae Lamiaceae Lamiaceae Apiaceae Lamiaceae Lamiaceae

W W W W W PH PH PH W W W W PH PH W W PH PH W W PH PH AH W W PH W PH PH W W W PH PH W W

+ + + + NA + + NA + + + + + NA + + + NA + + NA + NA + + + + NA NA NA + NA NA

+ NA NA + + + NA + + NA NA NA + NA NA + + NA NA NA NA NA NA NA + NA NA NA

41 28 21 41 21 21 14, 38 17, 21 37, 51, 52 35 33, 35, 50 33, 35 21 21 24 41, 46 21 21 21 28, 39 21 21 17 21 21 21 21 7 21 7 21 21 7 21 21 21

Paula & Pausas - J. Ecol. - Supplementary material: Appendix S1 - 4 Thymus vulgaris L. Trifolium campestre Schreb. Trifolium glomeratum L. Tuberaria guttata (L.) Fourr. Tuberaria lignosa (Sweet) Samp. Ulex europaeus L. Ulex parviflorus Pourr.

Lamiaceae Fabaceae Fabaceae Cistaceae Cistaceae Fabaceae Fabaceae

W AH AH AH W W W

NA + -

+ + + + + + +

6, 21 47 47 15 21, 44 29 3, 4, 5

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